David Slade

Artificial seeds for plant propagation

Abstract Artificial seeds consisting of tissue-culture produced embryos encased in a protective coating, will allow the economical mass propagation of elite plant varieties. They would also be a channel for new plant lines produced through biotechnological advances to be delivered directly to the greenhouse or field.

Alfalfa somatic embryo maturation and conversion to plants

Abstract Maturation of somatic embryos of alfalfa ( Medicago sativa L.) on medium containing abscisic acid (ABA) prior to planting increased plant formation from somatic embryos (‘conversion’) from 0% to 80% under growth chamber conditions. The embryos were planted directly into soil potting mix under non-sterile conditions without any exogenous nutrients. There was a positive correlation of increased conversion frequency and high embryo fresh/dry weights, dry matter content and starch levels when the somatic embryos were matured on ABA medium. Starch grains were present in the cotyledons and axis of somatic embryos that were ABA-matured and absent in embryos that did not receive the ABA treatment. ABA may be promoting somatic embryo storage reserve accumulation necessary for high conversion and vigor in the soil environment.

Maturation and greenhouse planting of alfalfa artificial seeds

SummaryConversion (plant production) was obtained from direct-planting alfalfa somatic embryos and encapsulated somatic embryos (artificial seeds) of alfalfa into a growth chamber and greenhouse. The embryos were planted in a commercial soil potting mix under nonsterile conditions in a manner similar to zygotic seed. Embryo maturation with abscisic acid (ABA), prior to planting, gave 48% conversion in soil under growth chamber conditions. Under greenhouse conditions, 64% conversion was obtained when humidified air was used to prevent soil surface drying. Previously, conversion in soil was between 0–6% without the ABA maturation treatment. The replacement of ABA with mannitol or combinations of mannitol and ABA during maturation resulted in lower conversion in the growth chamber than with ABA alone. ABA may be promoting the accumulation of embryo storage reserves such as proteins and carbohydrates for growth after planting in the soil environment.

FIELD PLANTING OF ALFALFA ARTIFICIAL SEEDS

SummaryEncapsulated somatic embryos (artificial seeds) and naked (uncoated) somatic embryos of alfalfa (Medicago sativa L.) were planted directly into the field to demonstrate the feasibility of using artificial seeds for direct sowing. Various row coverings that provided protection for the somatic embryos during conversion (plant formation) in the field and encapsulation methods were investigated. The highest conversion obtained in the field was 25% when naked somatic embryos were planted under the protective covering of inverted styrofoam cups. In comparison, 60% conversion was obtained when embryos were planted in potting mix in a growth chamber. Somatic embryos encapsulated by the thin-coat method converted at 23% under cups in the field and 40% in potting mix in the growth chamber. Naked somatic embryos had an average of 13 and 9% conversion in the field under plastic and cloth coverings, respectively, whereas encapsulated embryos converted at 5 and 14%, respectively. Direct-planted embryos (no row covering) converted at 1% in the field. Successful conversion of coated and naked somatic embryos planted in the field supports the concept of artificial seeds serving as a substitute for natural seeds.

Effect of organic acid pretreatment on the regeneration and development (conversion) of whole plants from callus cultures of alfalfa, Medicago sativa L.

Abstract Cultures of uninduced alfalfa callus have been pretreated with various organic acids for one subculture cycle prior to embryo induction with 2,4-dichlorophenoxyacetic acid (2,4-D) and regeneration on hormone-free medium. This culture pretreatment regime results in decreased callus growth, changes the morphology of the callus and causes an increase in embryo yield after induction and regeneration of the callus. The effective organic acids and their optimum concentrations are K+-citrate (20 mM), K+-malate (60 mM), K+-succinate (≤50 mM), K+-tartrate (70 mM) and K+-oxadate (10 mM). The effect of K+-citrate can be enhanced by the addition of amino acids such as proline or glutamine to the pretreatment medium. The embrys which form from pretreated callus have more normal morphology than embryos coming from untreated callus cells. A further effect of organic acid pretreatment is to increase the percentage of whole plants which develop (conversion) compared to control treatments. The efficiency of regeneration of callus to whole plants was determined for control and organic acid pretreated cultures. The results indicate that while callus growth rate is 44–64% less during organic acid pretreatment compared to the control, the overall efficiency of regeneration is increased by 88–320%. The effects on culture buffering and chelation apparently do not account for the organic acid pretreatment effect. Organic acid pretreatment alone does not satisfy the requirement for induction of callus with a strong auxin such as 2,4-D. The results are discussed with reference to physiological selection of callus which is competent to undergo embryogenesis.

Artificial seeds: A method for the encapsulation of somatic embryos

A detailed description of alfalfa (Medicago sativa L.) somatic embryogenesis and methods to produce alfalfa artificial seeds are described. These protocols provide directions for producing alfalfa artificial seeds that will convert into whole plants when planted in vitro or directly into soil.